SUMMARY/ABSTRACT Lung cancer is the leading cause of mortality in the US, affecting 160,000 patients annually with a five-year survival of 17%. The majority of lung cancers harbor KRAS mutations that overactivate the RAS kinase in the in the MAPK signaling pathway, RAS-RAF-MEK-ERK. Kinase inhibitors (KIs), which target key effectors of cancer signaling pathways, constitute a major strategy to treat KRAS-driven lung cancers. Potent MEK inhibitors exist that inhibit downstream ERK signaling in many tumors, but these also suppress signaling in normal cells so that their dosing is limited by toxicity, resulting in a narrow therapeutic index. The anti-tumor effects of MEK inhibitors are also diminished by the activation of the compensatory or parallel FGFR1 pathway, resulting in drug resistance and tumor resurgence. The co-administration of a second KI of the FGFR1 pathway can mitigate this resistance, but many such combinations of KI are highly toxic. Nanoscale drug delivery is a promising strategy to overcome the limitations of applying combination KI therapy to KRAS mutant lung cancer. We have discovered that interactions between charged dyes can stabilize diverse drug cargoes to form nanoparticles. These dyes have a sulfated surface that exhibits selective uptake by cell types with high CAV1 expression ? such as KRAS lung tumor endothelium. In Aim 1, we propose to investigate the mechanism of this CAV1 targeting and assess its potential to alter the pharmacokinetic and biodistribution of packaged drugs versus free drugs. We plan to use pharmacological inhibitors of various endocytosis pathways in endothelial cells in vitro to confirm that the primary mechanism of nanoparticle uptake is caveolin-mediated endocytosis. We will also use transwell assays to understand how CAV1 knock out in lung endothelial cells would alter the ability of nanoparticles to transcytose into the tumor environment. We will then conduct in vivo time lapse intravital microscopy of the lung tumor microenvironment in mice treated with nanoparticles to see in real-time how the nanoparticles target, interact, and cross the tumor endothelium. In Aim 2, we will assess the pharmacodynamics of nanoparticle delivered KIs versus free KIs as well as assess improvements to therapeutic index of nanoparticle-mediated RAS and FGFR pathway inhibition. Previously, we have found that nanoparticles resulted in long-term inhibition of pERK in tumors and significantly attenuated inhibition in the skin, demonstrating that this strategy may obviate a major side-effect of MEK inhibitors ? skin toxicities. Upon completion, this project will result in new findings that may produce novel therapies for the treatment of KRAS lung cancers. This project would establish that combination therapies could be delivered in nanoparticles that selectively target only the tumor microenvironment. This selective delivery would alter the dose limiting toxicities seen by these drugs upon systemic administration. Long term, these results can be used to inform future investigations in canine patients, IND-enabling studies, and clinical trials.